Laccase producing composition

ABSTRACT

The present invention provides a novel method for improving microbial laccase production, which relates to the field of microbial fermentation. The present invention is to add beta-carotene and other types of carotenoids, or microorganisms that produces carotenoids, or a mixture comprising carotenoids into a fermentation system during fermentation of  Pleurotus ferulae  and other higher fungi. The present invention can improve the laccase production 12 times more than before. With the advantage of a simple process and high yield, the present invention has a bright application prospect.

CROSS-REFERENCES AND RELATED APPLICATIONS

This application claims the benefit of priority to U.S. application Ser.No. 15/268,892 filed on Sep. 19, 2016, which claims Chinese ApplicationNo. 201610507284.2, entitled “A novel method for improving microbiallaccase production”, filed Jun. 30, 2016, all of which are hereinincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the field of microbial fermentation,which relates to a novel method for improving microbial laccaseproduction.

Description of the Related Art

Laccase (p-diphenoloxidase, EC 1.10.3.2) are a group of blue multicopperoxidases that are capable of oxidizing mono-, di- and polyphenols,aminophenols, methoxyphenols, aromatic amines and ascorbic acid. Varioussubstrates can be oxidized by laccase catalysis, which extends theindustrial application in pulp and paper industry, wastewater treatmentindustry, organic synthesis and so on.

Laccase is mostly produced from filamentous fungi, especiallybasidiomycetes and ascomycetes fungi. However, low laccase productionfrom ascomycetes and basidiomycetes could hardly meet the growing demandfor industrial utilization of laccase. Besides the bacteria, nutrients,and cultivation conditions, some kinds of stimulators affect the laccaseproduction by basidiomycetes and ascomycetes. The copper ions are one ofthe compositions of laccase activity center and an effective stimulatorfor laccase synthesis as well. Meanwhile, some small-molecules aromaticcompounds including guaiacol, veratryl alcohol, vanillin and cinnamicacid are also used as stimulators for improving laccase productionbecause of the structure similarly to the lignin. However, thesimulators are found to be usually very expensive, which lead to highercost in laccase production. Meanwhile, production with addition ofcopper ions or aromatic compounds induces pollution to the environment,and results to following detoxification treatment for culture, whichinfluences the industrialization of laccase application. A novel,economic, safe and efficient stimulator is necessary and will surely bean important researching direction for improving laccase production. Itmay also inject new energies into microbial fermentation and industrialproduction.

DETAILED DESCRIPTION

The first goal of the present invention is to provide a novel stimulatorfor microbial laccase production by a laccase-producing microorganism.

An active ingredient of the stimulator comprises carotenoid, and thestimulator comprises one or more types of the carotenoids.

The laccase-producing microorganism comprises a laccase-producingfilamentous fungi.

In one embodiment, the stimulator is β-carotene, lycopene, or both.

In one embodiment, the filamentous fungi comprises Pleurotus ferulae,Trametes versicolor, Pleurotus ostreatus, or Ganoderma lucidum.

In one embodiment, the active ingredient of the stimulator comprisescarotenoids, or one or more carotenoids-producing microorganisms, orextracts of carotenoids.

Another goal of the present invention is to provide a novel method forimproving microbial laccase production, and the method comprises addingthe stimulator during a fermentation process.

In one embodiment, the laccase-producing microorganism comprises afilamentous fungi which is able to produce laccase.

In one embodiment, the laccase-producing microorganism comprisesPleurotus ferulae, Trametes versicolor, Pleurotus ostreatus, orGanoderma lucidum.

In one embodiment, the stimulators comprises compounds of carotenoids,or a mixture comprising carotenoids; and the method comprises choosingor adjusting amount of the stimulator and time of adding the stimulatoraccording to a condition of the fermentation process; wherein themixture comprising carotenoids comprises an active carotenoid-producingmicroorganism, an inactivated carotenoid-producing microorganism, orextracts of carotenoids; and the active carotenoid-producingmicroorganism comprises Rhodotorula mucilaginosa, Phaffia rhodozyma,Sporidiobolus pararoseus, or Rhodotorula glutinis.

In one embodiment, the microorganism is bacteria or fungi thatfrequently used in this field; the cultivation method is common methodsused in this field.

In one embodiment, the amount of carotenoids is 0.1˜30 mg/L, thecarotene is dissolved in acetone, sterilized through hydrophobicmembrane to remove bacteria, and then evaporated to dryness throughrotary evaporator; the obtained substrates are then dissolved insterilized oil and added into fermentation medium.

The mixture comprising carotenoids comprises an activecarotenoid-producing microorganism or an inactivatedcarotenoid-producing microorganism, or extract comprising carotenoids.

In one embodiment, the method comprises cultivating an activecarotenoids-producing microorganism, and adding an appropriate amount ofthe carotenoids-producing microorganism into the fermentation processfor co-culture for a pre-determined amount of time.

In one embodiment, before being added into the fermentation system,inactivated carotenoids-producing organism is obtained through waterbath under 70° C. for 1 h.

In one embodiment, the extract comprising carotenoids is obtainedaccording to the following steps: (1) cells are disrupted by alternatefreezing and thawing, and then heated under 70° C. for 1 h, (2) aftercentrifugation, removing the supernatant and adding a certain volume ofvegetable oil to the precipitate; extract through slight oscillation for1 h and then oil extract is obtained and then been added into thefermentation system; the above operation is under a sterilizedenvironment.

The advantage of the present invention includes:

1. Multiple addition modes for stimulators. The addition mode in thepresent invention includes: straightly adding carotenoids, addingextract of carotenoids, adding microorganism that producing carotenoids,and adding treated microorganism that producing carotenoids.

2. Wide application. The stimulators in the present invention have theability of promoting a variety of microorganisms for laccase production.

3. Great promoting effect. The present invention provides a method forpromoting laccase production through adding carotenoids or yeast cellsthat producing carotenoids, which increases laccase activity overtwofold than that produced by Pleurotus ferulae; and 12 times than thatproduced by other higher fungi.

EXAMPLES

Enzyme Assay

The standard assay comprised following steps: A 1 mL reaction mixturecomprising 880 μl 0.1 mol·L⁻¹ acetic acid-sodium acetate (pH 4.5), 100μl ABTS stock (final assay concentration 1 mol·L⁻¹) and 20 μl dilutedenzyme (absorbance between 0.2˜0.8) is incubated at 30° C. for 5 min,final absorbance is spectrophotometrically monitored at 420 nm.

One unit of enzyme was defined as the amount of enzyme that oxidied of 1μmol ABTS per minute.

The enzyme activity is calculated according to the formula below:

${{Enzyme}\mspace{14mu}{activity}\mspace{14mu}(U)} = \frac{\left( {A_{2} - A_{1}} \right) \times 50 \times 10^{6}}{t \times ɛ}$

-   -   A₁—Absorbance of the blank;    -   A₂—Final absorbance;    -   t—Reaction time;    -   ε—molar absorptivity of ABTS, 3.6×10⁴ L·mol⁻¹·cm⁻¹.

Determination of β-carotene

(1) Standard solution preparation: A 2 mg standard β-carotene orlycopene was accurately weighted and dissolved in chloroform in a 10 mLbrown constant bottle, stored in the dark at −20° C.

(2) Sample preparation:

a) Determination of β-carotene or lycopene in culture broth: 80 mLculture broth and 80 mL chloroform were mixed and shaking-extracted for15 min, the resulting chloroform was concentrated to 10 mL in rotaryvacuum evaporator to prepare chloroform extract comprising β-carotene orlycopene;

b) Determination of β-carotene in Rhodotorula mucilaginosa: Rhodotorulamucilaginosa cell was disrupted by freeze-drying, 1 g cells was weightedand transferred to 5 mL EP tube, 2 mL chloroform is added andshaking-extracted for 15 min, the resulting chloroform was centrifuged,supernatant from which was volumed to 10 mL by adding chloroform toprepare chloroform extract comprising β-carotene.

c) Determination of β-carotene in co-culture broth: 150 mL culture brothwas centrifuged, cells collected from that was fully grinded,shaking-extracted for 15 min with 80 mL chloroform addition, theresulting chloroform solution was concentrated to 10 mL in rotary vacuumevaporator to prepare chloroform extract comprising β-carotene.

(3) LC-MS detection:

LC condition: the UPLC system equipped with an Waters Acquity PDAdetection and couples to WATERS MALDI SYNAPT Q-TOF MS was used fordetection. The extracted β-carotene is separated using Waters AcquityUPLC BEH C18 column (2.1 mm×100 mm) with a column temperature of 45° C.,a flow rate of 0.3 mL·mL⁻¹, an injection volume of 1 μL. The separationof β-carotene was achieved using a mobile phase consisting ofacetonitrile/iso-propyl alcohol, 10:90, v/v (solvent A) and acetonitrile(solvent B). The mass spectrometer was optimized in ESI+mode for thedetermination of β-carotene under following condition: capillaryvoltage: 3.5 kV, sample cone: 30 V; vaporizer temperature 330° C.; trapgas flow: 700 L·Hr⁻¹; collision energy: 6 eV; quality range: 100-1500;voltage detection 1800 V

(4) The content of β-carotene was calculated according to the formulabelow:

$m = \frac{m_{1} \times A_{2} \times V_{2} \times 10}{m_{2} \times A_{1} \times V_{1}}$

m: Content of β-carotene or lycopene in 10 mL sample (mg);

m₁: Content of β-carotene or lycopene in standard sample (mg);

m₂: Weight of sample (mg);

A₁: Peak area of standard sample;

A₂: Peak area of sample;

V₁: Weight of sample (μL);

V₂: Volume of sample (mL);

Example 1 Laccase Production Added with β-carotene and Lycopene

Liquid medium (g·L⁻¹): glucose 20, corn meal 10, wheat bran 10, K₂SO₄0.1742, pH 9.0; medium was used for seed culture and liquid fermentationfor P. ferulae.

Seed preparation: 2 pieces of P. ferulae with an area of 0.5 cm² weretransferred to 80 mL seed medium, incubated at 25° C. under 150 r·min⁻¹for 7 d.

Laccase fermentation: 3% (v/v) seed culture broth was inoculated into150 mL liquid medium, and then incubated at 25° C. under 150 r·min⁻¹ for7 d.

4 mg β-carotene and 4 mg lycopene was respectively weighted anddissolved in acetone, 0.22 μm hydrophobic membrane was used to removebacterial from the acetone solution. The sterile solution wastransferred to 50 mL tube and then evaporated to dryness through rotaryvacuum evaporator. The resulting dryness was collected and dissolved in1.0 sterile vegetable oil. The wherein said oil was added into 2-day P.ferulae fermentation system and then continued fomenting for another 5d. The fermentation without addition of β-carotene or lycopene was setas control. Content of β-carotene and lycopene were detected during thefermentation. Laccase was determined when the fermentation finished. Itwas shown that β-carotene concentration was 25.37 mg/L, 13.13 mg/L, 6.90mg/L, 1.66 mg/L, and 0 mg/L respectively from the initial day to the4^(th) day of the fermentation, the lycopene in culture broth from thosedays was 24.83 mg/L, 3.22 mg/L, 1.59 mg/L, 0.31 mg/L and 0 mg/L,respectively.

Example 2 Laccase Production Added With Rhodotorula mucilaginosa

YPD medium (g·L⁻¹): peptone 20, yeast extract 10, glucose 20, at naturepH.

Seed preparation: colony of R. mucilaginosa was picked and inoculatedinto 80 mL YPD medium, incubated at 30° C. under 200 r·min⁻¹ for 48 h.

P. ferulae was incubated according to Example 1.

Co-culture of P. ferulae and R. mucilaginosa: 3% (v/v) R. mucilaginosawas inoculated when P. ferulae had been incubated for 48 h, both of thestrains were then co-cultured for another 5 d. The P. ferulaefermentation without R. mucilaginosa addition was set as control.Laccase activity was determined when fermentation finished.Concentration of β-carotene was determined during fermentation process.The results show that content of β-carotene in culture broth was 0.0015mg/L, 0.0040 mg/L, and 0.013 mg/L respectively on the 1^(st), 3^(rd),and 5^(th) day of co-culture.

The laccase activity from fermentation with R. mucilaginosa addition was7630 U·L⁻¹, while laccase activity in control group was only 3500 U·L⁻¹.

Example 3 Laccase Production Added With Phaffia rhodozyma, Sporidioboluspararoseus or Rhodotorula glutinis

P. ferulae was incubated according to Example 1.

P. rhodozyma, S. pararoseus and R. glutinis were incubated according toExample 2

P. ferulae was coincubated with P. rhodozyma, S. pararoseus and R.glutinis were incubated according to Example 2.

Experimental group: inoculation amount of R. mucilaginosa is 1.25 mL,2.5 mL, 5 mL, and 10 mL respectively. Fermentation without R.mucilaginosa addition was set as control.

Laccase production of Pleurotus ferulae improved when inoculatet with S.pararoseus at an amount of 2.5 mL, 5 mL, and 10 mL. Besides, the laccaseactivity reached to peak at 10000 U·L⁻¹ under 2.5 mL inoculation amountof S. pararoseus. The content of β-carotene was 0.027 mg/L, 0.036 mg/L,and 0.040 mg/L in culture broth respectively from the 1^(st), 3^(rd),and 5^(th) day of co-culture.

The laccase activity achieved to 7161 U·L⁻¹ under 1.25 mL inoculationamount of R. glutinis when P. ferulae co-cultured with R. glutinis,while laccase activity was 3700 U·L⁻¹ in control group.

Example 4 Laccase Production Added With Heat-Treated Rhodotorulamucilaginosa

P. ferulae was cultivated according to Example 1.

R. mucilaginosa was cultivated according to Example 2, and thenrespectively treated under 55° C., 60° C., 65° C., and 70° C. water-bathfor 1 h.

High-temperature sterilized group (HTS group): R. mucilaginosa treatedunder 121° C. for 20 min.

Control group 1: fermentation without R. mucilaginosa addition.

Control group 2: fermentation with untreated R. mucilaginosa.

Spread plate was employed to evaluate the effect of different conditionof heat treatment. It was shown that, colony number of R. mucilaginosadecreased for over 50% after heat-treated at 55° C. for 1 h. Highertemperature led to lower number of colonies. The effect of treatmentunder 70° C. or above for 1 h was the same with the effect of HTS group,both of which had a completely sterilization.

Cells of R. mucilaginosa from above groups with wet weight of 1.5 g, 3.0g, 4.5 g, 6 g were added into P. ferulae fermentation system after 48-hincubation, continued fermenting for another 5 d, laccase activity weredetermined. The resulting culture broth involved with inactive R.mucilaginosa (treated under 70° C.) showed a high laccase activity.Besides, the laccase activity increased with the addition of cells.Laccase activity in broth with 6 g R. mucilaginosa addition achieved7260 U·L⁻¹.

Example 5 Laccase Production Added With Extract Comprising β-carotene

P. ferulae was incubated according to Example 1.

R. mucilaginosa was incubated according to Example 2.

Cells of R. mucilaginosa was collected under aseptic condition. Wetweight of 4 g cells mixed with sterilize water was disrupted throughrepeated frozen and thawed, and then 70° C. treated for 1 h,centrifugation was employed to collect the precipitate from thesolution. Sterilize vegetable oil with precipitate was mixed, extractedcombined with shaking for 1 h to obtain the oil extract of R.mucilaginosa. The extract was added into 2-day fermentation system of P.ferulae. The fermentation was then continued for another 5 days andlaccase in the culture broth was determined. Fermentation added withequivalent vegetable oil was set as control.

It was shown that, laccase production improved 35% after adding with 3mL extract oil, which achieved a laccase activity of 4850 U·L⁻¹.

Example 6 Laccase Production Added With Rhodotorula mucilaginosa

Higher fungi, such as T. versicolor, P. ostreatus, and G. lucidum wascoincubated with R. mucilaginosa.

R. mucilaginosa was cultivated according to Example 2.

Medium for T. versicolor, P. ostreatus, and G. lucidum was: wheat bran1%, corn meal 1%, glucose 2%, MgSO₄.7H₂O 0.2%, and KH₂PO₄ 0.3%.

Cultivation condition for T. versicolor, P. ostreatus, and G. lucidum:inoculation volume 3%; incubation lasted 5 d, 6 d, and 8 d for T.versicolor, P. ostreatus, and G. lucidum, respectively. R. mucilaginosawas inoculated at a volume of 0, 1.5%, 3%, 6% and laccase activitieswere determined after 5-day co-culture.

As the results, laccase activity was increased in fungi fermentationinvolved with R. mucilaginosa. The laccase produced by T. versicolor was1873 U·L⁻¹, it achieved 3797 U·L⁻¹ with 6% R. mucilaginosa participatedin fermentation. The laccase produced by P. ostreatus was 1147 U·L³¹ ¹with 3% R. mucilaginosa participated in fermentation, which was six foldof that in fermentation without R. mucilaginosa addition. The laccaseproduced by G. lucidum was only 80.3 U·L⁻¹, with increasing of R.mucilaginosa addition, it achieved 982.8 U·L⁻¹, which improved 12 timesthan initial production.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables,appendices, patents, patent applications and publications, referred toabove, are hereby incorporated by reference.

What is claimed is:
 1. A composition comprising a Sporidioboluspararoseus and a carotenoid, wherein the Sporidiobolus pararoseus andthe carotenoid improve laccase production.
 2. The composition of claim1, comprising β-carotene, lycopene or a mixture thereof.
 3. A method ofimproving laccase production, comprising adding the composition of claim1 during a fermentation process.
 4. The method of claim 3, furthercomprising choosing or adjusting amount of the composition and time ofadding the composition according to a condition of the fermentationprocess.
 5. The method of claim 3, further comprising cultivatingSporidiobolus pararoseus, and adding an appropriate amount of theSporidiobolus pararoseus into the fermentation process for co-culturefor a pre-determined amount of time.
 6. A method of improving laccaseproduction, comprising: adding a composition comprising a Sporidioboluspararoseus microorganism and a carotenoid during a fermentation process.7. The method of claim 6, further comprising choosing or adjustingamount of the composition and time of adding the composition accordingto a condition of the fermentation process.
 8. The method of claim 6,further comprising cultivating Sporidiobolus pararoseus, and adding anappropriate amount of the Sporidiobolus pararoseus into the fermentationprocess for co-culture for a pre-determined amount of time.